PROJECT SUMMARY Epigenetics is the study of changes in gene expression or phenotypes that are not the result of changes in DNA sequence. RNA has emerged as an important informational vector directing many epigenetic processes. Additionally, for most of the last century it was widely believed that (unlike genetic information) epigenetic information did not pass across generational boundaries. In other words, epigenetic information was erased each and every generation such that offspring began life with a blank epigenetic slate. It is now known that this is not always the case. Many examples of the trans-generational transfer of epigenetic information have now been documented. The inheritance of epigenetic information for more than one generation is termed transgenerational epigenetic inheritance (TEI). Non-coding RNAs and, in particular, small non-coding RNAs such as piRNAs, miRNAs, siRNAs, and tRNAs have now been linked to TEI in plants, worms, insects, and mammals. Thus, small non-coding RNAs are important informational vectors for TEI in many eukaryotes. In most eukaryotes, dsRNA induces gene silencing (RNAi). We have used RNAi in C. elegans to identify factors that couple small non-coding RNAs to transcriptional regulation. Recently, these studies led us to discover a new type of silencing RNA that we term the pUG RNA. Amazingly, progeny of C. elegans subjected to RNAi inherit the ability to silence RNAi-targeted genes for many (5-10) generations (termed RNAi inheritance). Thus, RNAi inheritance in C. elegans is a particular robust example of RNA-directed TEI. We are also using RNAi inheritance in C. elegans as a model system to explore the mechanistic underpinnings of RNA-directed TEI in animals. We are using genetic screens to identify cellular factors required for promoting and limiting TEI and biochemical and cell biological approaches to explore how these factors drive TEI. Finally, we are also using this system to explore why animals have TEI systems in the first place. The evolutionarily conserved connections between non-coding RNAs and TEI processes in many different species suggests that the work we are doing in C. elegans may lead to fundamental insights into mechanisms of TEI, which will be applicable to eukaryotes in general. The mis-regulation of epigenetic pathways is known to contribute to the etiology of dozens of human diseases, including cancer. Our proposed work will likely increase our understanding of how RNA reprograms epigenetic states and, therefore, may help us understand and, possibly, treat these diseases. Additionally, the question of whether or not people can inherit epigenetic information from their parents is the subject of intense scientific debate. If people can indeed inherit epigenetic information from their parents then it stands to reason they could inherit the wrong epigenetic information, which might predispose to disease. Our work exploring mechanisms of RNA-directed TEI may make it possible to influence TEI pathways in people to mitigate disease.